БИОЛОГИЧЕСКИЕ МЕМБРАНЫ, 2012, том 29, № 1-2, с. 102-113

УДК 577.352.4


© 2012 B. I. Khodorov1*, M. M. Mikhailova1, A. P. Bolshakov2, A. M. Sarin1, 3, E. G. Sorokina3, S. A. Rozhnev1, V. G. Pinelis3

institute of General Pathology and Pathophysiology, Russian Academy of Medical Sciences, ul. Baltiiskaya, 8, Moscow, 125315Russia; Tel: +7(499)134-1445, Fax: +7(495)151-1726;

*e-mail: boriskhodorov@mail.ru 2Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, ul. Butlerova, 5a, Moscow, 117485 Russia 3Scientific Center of Children's Health, Russian Academy of Medical Sciences, Lomonosovskii pr. 2/62, 119991, Moscow, Russia Received 20.09.2011

Cultured cerebellar granule cells were co-loaded with Ca2+-sensitive dye fura-2FF and rhodamine-123 sensitive to changes in the mitochondrial potential (A^m). A 60-min incubation of cells in glucose-free solution containing 2-deoxy-D-glucose (DG) induced a slow developing mitochondrial depolarization (sMD) without appreciable changes in basal [Ca2+]i. This sMD was insensitive to a removal of external Ca2+ or to the NMDA channels blocker memantine but could be readily suppressed by oligomycin due to inhibition of the inward proton current through the Fo channel of mitochondrial ATP synthase. In resting cells glucose deprivation caused a progressive decrease in mitochondrial NADH content ([NADH]), which strikingly enhanced the ability of glutamate to induce a delayed Ca2+ deregulation (DCD) associated with a profound mitochondrial depolarization. In glucose-containing medium this DCD appeared in young cells (usually 6—8 DIV) after a prolonged latent period (lag phase). Substitution of glucose by DG led to a dramatic shortening of this lag phase, associated with a critical decrease in [NADH] in most neurons. Addition of pyruvate or lactate to DG-con-taining solution prevented the sMD and [NADH] decrease in resting cells and greatly diminished the number of cells exhibiting glutamate-induced DCD in glucose-free medium. Measurement of intracellular ATP level ([ATP]) in experiments on sister cells showed that glucose deprivation decreased [ATP] in resting cells and considerably deepened the fall of [ATP] caused by glutamate. This decrease in [ATP] was only slightly attenuated by pyruvate and lactate, despite their ability to prevent the shortening of lag phase preceding the DCD appearance under these conditions. Simultaneous monitoring of cytosolic ATP concentration ([ATP]c) and A^m changes in individual CGC expressing fluorescent ATP sensor (AT1.03) revealed that inhibition of either mitochondrial respiration or glycolysis caused a relatively small decrease in [ATP]c and A^m. Complete blockade of ATP synthesis in resting CGC with oligomycin in glucose-free DG-containing buffer caused fast ATP depletion and mitochondrial repolarization, indicating that mitochondrial respiratory chain still possess a reserve fuel to support A^m despite inhibition of glycolysis. The data obtained suggest that the extraordinary enhancement of glutamate-induced deterioration in Ca2+ homeostasis caused by glucose deprivation in brain neurons is mainly determined by NADH depletion.

Keywords: cerebellar granule cells, 2-deoxy-^D-glucose, delayed calcium deregulation, pyruvate.

Deregulation of neuronal Ca2+ homeostasis following overstimulation of glutamate (Glu) receptors is considered as an experimental model of excitotoxic brain injury observed in stroke and some neurological disorders (for reviews, see [1, 2]). Over the last decade, numerous studies were performed to clarify the mechanisms of the Glu-induced Ca2+ overload and a concurrent strong mitochondrial depolarization (MD) in various types of mammalian central neurons (for review, see [2, 3]). However, despite a considerable progress, the exact mechanism of the Glu-induced MD and its coupling to [Ca2+] deregulation is yet not deciphered. Earlier it has been shown that Glu-in-

duced disturbance of [Ca2+]i homeostasis strongly depends on the age of the culture [4—6]. Thus, a prolonged (10—15 min) Glu challenge produces a biphasic MD in mature cultured hippocampal neurons (>11 days in vitro). The secondary profound MD is associated with a synchronous [Ca2+]i elevation (so-called delayed [Ca2+]i deregulation, or DCD), which in many cells remains at high plateau level far beyond the Glu removal [5]. In contrast, the same Glu challenge evokes in younger cultured neurons (6—8 days in vitro) a very small and readily reversible MD associated with a small [Ca2+]i increase [5, 7]. The secondary increase in [Ca2+]i and MD occurs in most young cerebellar

granule cells (CGC) only 10—60 min after the Glu application (for review, see [1]). It has been shown that inhibition of mitochondrial ATP synthesis by oligo-mycin prior to DCD development has no impact on Glu-induced changes in [Ca2+] but slightly increases mitochondrial potential (A¥m) in young CGC [1, 8] and hippocampal neurons [5, 9]. Therefore, to clarify the reasons of high resistance of young cells to a toxic Glu challenge, we examined the effect of glycolysis inhibition on the glutamate-induced changes in [Ca2+]j, A¥m, and NADH content ([NADH]) in young cultured neurons. Blockade of glycolysis was performed by replacement of glucose with 2-deoxy-^-glucose (DG), which is known to compete with glucose for phosphorylation by hexokinase [10, 11]. DG suppresses the cytosolic production of both ATP and pyruvate (Pyr) (see [12] and references therein). Pyr links glycolysis with the mitochondrial tricarboxilic acid (TCA) cycle, which fuels respiratory chain maintaining A¥m.

Earlier [13, 14] it has been shown that glucose deprivation strikingly diminishes the ability of young CGC and cortical neurons to withstand the glutamate-induced deterioration of calcium homeostasis. In our experiments a dramatic shortening of the lag phase of DCD and of the MD emergence was observed. To clarify the reason for these changes, we examined the impact of a prolonged glucose deprivation on the bioenergetics state of nerve cells. It has been found that the replacement of glucose with DG induces slow mitochondrial depolarization (sMD), progressive decrease in the NADH and ATP content, and dramatic acceleration of the DCD development. The respiratory substrates pyruvate (Pyr) or lactate (Lac) prevented all these changes. These observations point to a common origin of DCD and sMD: deficiency of pyruvate. Similar effect was exerted by Pyr and Lac on the sensitivity of nerve cells to glutamate in glucose-free buffer.

The data reported here suggest that a progressive decrease in mitochondrial [NADH] to a critical level during a prolonged glucose deprivation is the main cause of a dramatic acceleration of the Glu-induced DCD (reduction of the lag phase) in CGC.


Preparation of cell cultures. Primary cultures of cerebellar granule neurones were prepared from 7-8-day-old Wistar rat pups as described earlier [15]. Cell suspensions (106 cells/ml) were plated on 25-mm PEI-coated glass coverslips or 35 mm MatTeck plastic dishes (MatTeck Corporation, USA) with coverslips attached to the 14-mm well in the dish bottom. Cultured neurons were maintained in CO2-incubator and used for fluorescence microscopy measurements on day 6 to 9 in vitro (6-9 DIV).

Cell culture transfection with plasmids. To monitor single-cell cytosolic [ATP] ([ATP]c) changes, the neurons were transfected with a plasmid encoding the ATP sensor AT1.03 [16], using 2 |l Lipofectamine-2000 and 1 |g DNA per 250 |l of medium (50 |l OptiMem + 200 |l of the cell transfection medium). Fluorescence analyses were carried out 2-4 days after the transfection.

Single-cell fluorescence imaging. To monitor [Ca2+]j changes, the neurons were loaded with fluorescent indicators Fura-2FF by addition to the cultures of the acetoximethyl ether of the indicator (Fura-2FF/AM, 1-2 |M, 50 min at 26-28°C). Membrane-permeable fluorescence cationic dye rhodamine 123 (Rh123, 2.5 |g/ml, 15 min) or tetramethylrhodamine methyl ether (TMRM, 40 nM, permanently in all buffers) were employed for the A¥m measurements. Dye load and cell imaging were performed at 26-28°C in buffered saline (if not specified otherwise) (mM): 130 NaCl, 5 KCl, 2 CaCl2, 1 MgSO4, 20 HEPES, 5 glucose; pH 7.4. In nominally Ca2+-free buffer Ca2+ was omitted and 0.1 mM Ca2+ chelator EGTA, and 2 mM MgSO4 were added. In glucose-free buffer glucose was substituted by 2-deoxy-^-glucose (DG-buff-er). Images were obtained employing epifluorescence inverted microscopes Axiovert 200 (Ziess) or Olympus IX70 (objectives 40x/1.35 oil or 20x/0.70). For simultaneous monitoring of autofuorescence of NAD(P)H (excitation, 365 ± 10 nm; emission, 450 ± 20 nm) and signals of potential-sensitive dyes or Ca2+ indicators a triple-band beam-splitter with maximum reflections at 440, 490 and 570 nm (TFT 440-490-570) was employed. A similar beam-splitter was used for simultaneous monitoring AT1.03 fluorescence and TMRM signals. AT1.03 fluorescence was excited using 440 ± 10 and 485 ± 15-nm filters, and emission was registered using 525 ± 20 nm filter. TMRM was excited with 565 ± 12 nm and registered with long-pass 610LP filter. All filters and beam-splitters were from Omega (USA). Images were acquired by Photometrics, Cool-Snap HQ camera and collected and analyzed using the Metafluor 6.1 software (Molecular Devices, USA).

ATP assay in cultured neurons. For measurements of the ATP content in the neuronal cell population ([ATP]), granule cells were plated on 24-well plates. In 7-8 days, the cells were washed twice with HBSS at room temperature and exposed to various treatments. After the exposure, ATP was extracted using ice-cold 2% trichloracetic acid supplemented with 2 mM EDTA. Cellular extract was neutralized by a 3 M KOH/1.5 M Tris solution and centrifuged at 3000 rpm. Immediately after that the ATP content

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